Srs2 Plays a Critical Role in Reversible G2 Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G₂ arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G₂ arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G₂ arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.DNA damage occurs frequently in all organisms as a consequence of both endogenous metabolic processes and exogenous DNA-damaging agents. In nature, the steady-state level of DNA damage is usually very low. However, chronic low-level DNA damage can lead to age-related genome instability as a consequence of the accumulation of DNA damage (12, 27). Increasing evidence implicates DNA damage-related replication stress in genome instability (7, 21). Replication stress occurs when an active fork encounters DNA lesions or proteins tightly bound to DNA. These obstacles pose a threat to the integrity of the replication fork and are thus a potential source of genome instability, which can contribute to tumorigenesis and aging in humans (4, 11). Confronted with this risk, cells have developed fundamental DNA damage response mechanisms in order to faithfully complete DNA replication (8).In budding yeast Saccharomyces cerevisiae, the Rad6-dependent postreplication repair (PRR) pathway is subdivided into three subpathways, which allow replication to resume by bypassing the lesion without repairing the damage (3, 22, 33). Translesion synthesis (TLS) pathways dependent on the DNA polymerases eta and zeta promote error-free or mutagenic bypass depending on the DNA lesion and are activated upon monoubiquitination of proliferating cell nuclear antigen (PCNA) at Lys164 (K164) (5, 16, 37). The Rad5 (E3) and Ubc13 (E2)/Mms2 (E2 variant)-dependent pathway promotes error-free bypass by template switching and is activated by polyubiquitination of PCNA via a Lys63-linked ubiquitin chain (16, 38, 41). It remains mechanistically unclear how polyubiquitinated PCNA promotes template switching at the molecular level. In addition to its ubiquitin E3 activity, Rad5 also has a helicase domain and was recently shown to unwind and reanneal fork structures in vitro (6). This led to the proposal that Rad5 helicase activity is required at replication forks to promote fork regression and subsequent template switching. It is possible that PCNA polyubiquitination acts to facilitate Rad5-dependent template switching by inhibiting monoubiquitination-dependent TLS activity and/or by recruiting alternative proteins to the fork.In addition to modification by ubiquitin, PCNA can also be sumoylated on Lys164 by the SUMO E3 ligase Siz1 (16). A second sumoylation site, Lys127, is also targeted by an alternative SUMO E3 ligase, Siz2, albeit with lower efficiency (16, 30). PCNA SUMO modification results in recruitment of the Srs2 helicase and subsequent inhibition of Rad51-dependent recombination events (29, 32). The modification can therefore allow the replicative bypass of lesions by promoting the RAD6 pathway. Srs2 is known to act as an antirecombinase by eliminating recombination intermediates. This can occur independently of PCNA sumoylation, and when srs2Δ cells are UV irradiated or other antirecombinases, such as Sgs1, are concomitantly deleted, toxic recombination structures accumulate (1, 10). Such genetic data are consistent with the ability of Srs2 to disassemble the Rad51 nucleoprotein filaments formed on single-stranded DNA (ssDNA) in vitro (20, 40). In addition to directly inhibiting homologous recombination (HR), Srs2 is also involved in regulating HR outcomes to not produce crossover recombinants in the mitotic cell cycle (18, 34, 35).The UV spectrum present in sunlight is a primary environmental cause of exogenous DNA damage. Sunlight is a potent and ubiquitous carcinogen responsible for much of the skin cancer in humans (17). In the natural environment, organisms are exposed to chronic low-dose UV light (CLUV), as opposed to the acute high doses commonly used in laboratory experiments. Hence, understanding the cellular response to CLUV exposure is an important approach complementary to the more traditional laboratory approaches for clarifying the biological significance of specific DNA damage response pathways. A recently developed experimental assay for the analysis of CLUV-induced DNA damage responses was used to show that the PCNA polyubiquitination-dependent error-free PRR pathway plays a critical role in tolerance of CLUV exposure by preventing the generation of excessive ssDNA when replication forks arrest, thus suppressing counterproductive checkpoint activation (13).Mutants of SRS2 were first isolated by their ability to suppress the radiation sensitivity of rad6 and rad18 mutants (defective in PRR) by a mechanism that requires a functional HR pathway (23, 36). In this study, we analyzed the function of Srs2 in CLUV-exposed PRR-deficient cells. We established that Srs2 acts in conjunction with SUMO-modified PCNA to lower the threshold for checkpoint activation and maintenance by suppressing the function of HR in rad18Δ cells exposed to CLUV. We also showed that Srs2 is separately involved in an HR-dependent recovery process following cessation of CLUV exposure and that this second role for Srs2, unlike its primary role in checkpoint activation and maintenance, is regulated by CDK1-dependent phosphorylation. Thus, Srs2 is involved in both CLUV-induced checkpoint-mediated arrest and recovery from CLUV exposure in PRR-deficient cells, and these two functions, while both involving HR, are separable and thus independent.     

Evidence for Mucin-Like Glycoproteins That Tether Sporozoites of Cryptosporidium parvum to the Inner Surface of the Oocyst Wall

Cryptosporidium parvum oocysts, which are spread by the fecal-oral route, have a single, multilayered wall that surrounds four sporozoites, the invasive form. The C. parvum oocyst wall is labeled by the Maclura pomifera agglutinin (MPA), which binds GalNAc, and the C. parvum wall contains at least two unique proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) identified by monoclonal antibodies. C. parvum sporozoites have on their surface multiple mucin-like glycoproteins with Ser- and Thr-rich repeats (e.g., gp40 and gp900). Here we used ruthenium red staining and electron microscopy to demonstrate fibrils, which appear to attach or tether sporozoites to the inner surface of the C. parvum oocyst wall. When disconnected from the sporozoites, some of these fibrillar tethers appear to collapse into globules on the inner surface of oocyst walls. The most abundant proteins of purified oocyst walls, which are missing the tethers and outer veil, were COWP1, COWP6, and COWP8, while COWP2, COWP3, and COWP4 were present in trace amounts. In contrast, MPA affinity-purified glycoproteins from C. parvum oocysts, which are composed of walls and sporozoites, included previously identified mucin-like glycoproteins, a GalNAc-binding lectin, a Ser protease inhibitor, and several novel glycoproteins (C. parvum MPA affinity-purified glycoprotein 1 [CpMPA1] to CpMPA4). By immunoelectron microscopy (immuno-EM), we localized mucin-like glycoproteins (gp40 and gp900) to the ruthenium red-stained fibrils on the inner surface wall of oocysts, while antibodies to the O-linked GalNAc on glycoproteins were localized to the globules. These results suggest that mucin-like glycoproteins, which are associated with the sporozoite surface, may contribute to fibrils and/or globules that tether sporozoites to the inner surface of oocyst walls.Cryptosporidium parvum and the related species Cryptosporidium hominis are apicomplexan parasites, which are spread by the fecal-oral route in contaminated water and cause diarrhea, particularly in immunocompromised hosts (1, 12, 39, 47). The infectious and diagnostic form of C. parvum is the oocyst, which has a single, multilayered, spherical wall that surrounds four sporozoites, the invasive forms (14, 27, 31). The outermost layer of the C. parvum oocyst wall is most often absent from electron micrographs, as it is labile to bleach used to remove contaminating bacteria from C. parvum oocysts (27). We will refer to this layer as the outer veil, which is the term used for a structure with an identical appearance on the surface of the oocyst wall of another apicomplexan parasite, Toxoplasma gondii (10). At the center of the C. parvum oocyst wall is a protease-resistant and rigid bilayer that contains GalNAc (5, 23, 43). When excysting sporozoites break through the oocyst wall, the broken edges of this bilayer curl in, while the overall shape of the oocyst wall remains spherical.The inner, moderately electron-dense layer of the C. parvum oocyst wall is where the Cryptosporidium oocyst wall proteins (Cryptosporidium oocyst wall protein 1 [COWP1] and COWP8) have been localized with monoclonal antibodies (4, 20, 28, 32). COWPs, which have homologues in Toxoplasma, are a family of nine proteins that contain polymorphic Cys-rich and His-rich repeats (37, 46). Finally, on the inner surface of C. parvum oocyst walls are knob-like structures, which cross-react with an anti-oocyst monoclonal antibody (11).Like other apicomplexa (e.g., Toxoplasma and Plasmodium), sporozoites of C. parvum are slender, move by gliding motility, and release adhesins from apical organelles when they invade host epithelial cells (1, 8, 12, 39). Unlike other apicomplexa, C. parvum parasites are missing a chloroplast-derived organelle called the apicoplast (1, 47, 49). C. parvum sporozoites have on their surface unique mucin-like glycoproteins, which contain Ser- and Thr-rich repeats that are polymorphic and may be modified by O-linked GalNAc (4-7, 21, 25, 26, 30, 32, 34, 35, 43, 45). These C. parvum mucins, which are highly immunogenic and are potentially important vaccine candidates, include gp900 and gp40/gp15 (4, 6, 7, 25, 26). gp40/gp15 is cleaved by furin-like proteases into two peptides (gp40 and gp15), each of which is antigenic (42). gp900, gp40, and gp15 are shed from the surface of the C. parvum sporozoites during gliding motility (4, 7, 35).The studies presented here began with electron microscopic observations of C. parvum oocysts stained with ruthenium red (23), which revealed novel fibrils or tethers that extend radially from the inner surface of the oocyst wall to the outer surface of sporozoites. We hypothesized that at least some of these fibrillar tethers might be the antigenic mucins, which are abundant on the surface of C. parvum sporozoites. To test this hypothesis, we used mass spectroscopy to identify oocyst wall proteins and sporozoite glycoproteins and used deconvolving and immunoelectron microscopy (immuno-EM) with lectins and anti-C. parvum antibodies to directly label the tethers.     

The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.DNA replication must be highly coordinated with chromatin assembly and cell division for correct propagation of genetic information and cell survival. Errors arising during DNA replication are corrected through the functions of numerous pathways including checkpoints and a diversity of DNA repair mechanisms (32, 33, 35). However, in the absence of these critical cellular responses, replication errors can lead to the accumulation of mutations and gross chromosomal rearrangements (GCRs) as well as chromosome loss, a condition generally termed genomic instability (33). Genome instability is a hallmark of many cancers as well as other human diseases (24). There are many mechanisms by which GCRs can arise, and over the last few years numerous genes and pathways have been implicated in playing a role in the suppression of GCRs in Saccharomyces cerevisiae and in some cases in the etiology of cancer (27, 28, 33, 39-47, 51, 53, 56, 58, 60), including S. cerevisiae ASF1, which encodes the main subunit of the replication coupling assembly factor (37, 62).Asf1 is involved in the deposition of histones H3 and H4 onto newly synthesized DNA during DNA replication and repair (62), and correspondingly, asf1 mutants are sensitive to chronic treatment with DNA-damaging agents (2, 30, 62). However, asf1 mutants do not appear to be repair defective and can recover from acute treatment with at least some DNA-damaging agents (2, 8, 30, 31, 54), properties similar to those described for rad9 mutants (68). In the absence of Asf1, both the DNA damage and replication checkpoints become activated during normal cell growth, and in the absence of checkpoint execution, there is a further increase in checkpoint activation in asf1 mutants (30, 46, 54). It has been suggested that asf1 mutants are defective for checkpoint shutoff and that this might account for the increased steady-state levels of checkpoint activation seen in asf1 mutants (8); however, another study has shown that asf1 mutants are not defective for checkpoint shutoff and that in fact Asf1 and the chromatin assembly factor I (CAF-I) complex act redundantly or cooperate in checkpoint shutoff (31). Furthermore, Asf1 might be involved in proper activation of the Rad53 checkpoint protein, as Asf1 physically interacts with Rad53 and this interaction is abrogated in response to exogenous DNA damage (15, 26); however, the physiological relevance of this interaction is unclear. Asf1 is also required for K56 acetylation of histone H3 by Rtt109, and both rtt109 mutants and histone H3 variants that cannot be acetylated (38) share many of the properties of asf1 mutants, suggesting that at least some of the requirement for Asf1 in response to DNA damage is mediated through Rtt109 (11, 14, 22, 61). Subsequent studies of checkpoint activation in asf1 mutants have led to the hypothesis that replication coupling assembly factor defects result in destabilization of replication forks which are then recognized by the replication checkpoint and stabilized, suggesting that the destabilized replication forks account for both the increased GCRs and increased checkpoint activation seen in asf1 mutants (30). This hypothesis is supported by other recent studies implicating Asf1 in the processing of stalled replication forks (16, 57). This role appears to be independent of CAF-I, which can cooperate with Asf1 in chromatin assembly (63). Asf1 has also been shown to function in disassembly of chromatin, suggesting other possibilities for the mechanism of action of Asf1 at the replication fork (1, 2, 34). Thus, while Asf1 is thought to be involved in progression of the replication fork, both the mechanism of action and the factors that cooperate with Asf1 in this process remain obscure.Stalled replication forks, particularly those that stall at sites of DNA damage, can be processed by homologous recombination (HR) (6) or by a mechanism known as postreplication repair (PRR) (reviewed in reference 67). There are two PRR pathways, an error-prone pathway involving translesion synthesis (TLS) by lower-fidelity polymerases and an error-free pathway thought to involve template switching (TS) (67). In S. cerevisiae, the PRR pathways are under the control of the RAD6 epistasis group (64). The error-prone pathway depends on monoubiquitination of proliferating cell nuclear antigen (PCNA) on K164 by Rad6 (an E2 ubiquitin-conjugating enzyme) by Rad18 (E3 ubiquitin ligase) (23). This results in replacement of the replicative DNA polymerase with nonessential TLS DNA polymerases, such as REV3/REV7-encoded DNA polymerase ζ (polζ) and RAD30-encoded DNA polη, which can bypass different types of replication-blocking damage (67). The error-free pathway is controlled by Rad5 (E3) and a complex consisting of Ubc13 and Mms2 (E2 and E2 variant, respectively), which add a K63-linked polyubiquitin chain to monoubiquitinated PCNA, leading to TS to the undamaged nascent sister chromatid (4, 25, 65). Furthermore, in addition to modification with ubiquitin, K164 of PCNA can also be sumoylated by Siz1, resulting in subsequent recruitment of the Srs2 helicase and inhibition of deleterious Rad51-dependent recombination events (50, 52, 55), although it is currently unclear if these are competing PCNA modifications or if both can exist on different subunits in the same PCNA trimer. A separate branch of the Rad6 pathway involving the E3 ligase Bre1 monoubiquitinates the histone H2B (29, 69) as well as Swd2 (66), which stimulates Set1-dependent methylation of K4 and Dot1-dependent methylation of K79 of histone H3 (48, 49, 66). Subsequently, K79-methylated H3 recruits Rad9 and activates the Rad53 checkpoint (19, 70). Activation of Rad53 is also bolstered by Rad6-Rad18-dependent ubiquitination of Rad17, which is part of the 9-1-1 complex that functions upstream in the checkpoint pathway (17). Finally, Rad6 complexes with the E3 Ubr1, which mediates protein degradation by the N-end rule pathway (13).Due to the role of the PRR pathways at stalled replication forks and a recent study implicating the Rad6 pathway in the suppression of GCRs (39), we examined the relationship between these ubiquitination and sumoylation pathways and the Asf1 pathway in order to gain additional insights into the function of Asf1 during DNA replication and repair. Our findings suggest that Asf1 has multiple functions that prevent replication damage or act in the cellular responses to replication damage and that these functions are modified by and interact with the PRR pathways. The TLS PRR pathway does not appear to be involved, and both a Dun1-dependent replication checkpoint and HR are important for preventing the deleterious effects of PRR and Asf1 pathway defects. We hypothesize that this newly observed cooperation between Asf1 and the PRR pathways may be required for resolving stalled replication forks, leading to suppression of GCRs and successful DNA replication.     

Two Fundamentally Distinct PCNA Interaction Peptides Contribute to Chromatin Assembly Factor 1 Function

Chromatin assembly factor 1 (CAF-1) deposits histones H3 and H4 rapidly behind replication forks through an interaction with the proliferating cell nuclear antigen (PCNA), a DNA polymerase processivity factor that also binds to a number of replication enzymes and other proteins that act on nascent DNA. The mechanisms that enable CAF-1 and other PCNA-binding proteins to function harmoniously at the replication fork are poorly understood. Here we report that the large subunit of human CAF-1 (p150) contains two distinct PCNA interaction peptides (PIPs). The N-terminal PIP binds strongly to PCNA in vitro but, surprisingly, is dispensable for nucleosome assembly and only makes a modest contribution to targeting p150 to DNA replication foci in vivo. In contrast, the internal PIP (PIP2) lacks one of the highly conserved residues of canonical PIPs and binds weakly to PCNA. Surprisingly, PIP2 is essential for nucleosome assembly during DNA replication in vitro and plays a major role in targeting p150 to sites of DNA replication. Unlike canonical PIPs, such as that of p21, the two p150 PIPs are capable of preferentially inhibiting nucleosome assembly, rather than DNA synthesis, suggesting that intrinsic features of these peptides are part of the mechanism that enables CAF-1 to function behind replication forks without interfering with other PCNA-mediated processes.Eukaryotic cells in S phase not only have to replicate their entire genome but also faithfully reproduce preexisting chromatin structures onto the two nascent chromatids. The duplication of chromatin structures during DNA replication is a challenging task for eukaryotic cells. Newly synthesized histones are deposited very rapidly behind replication forks (150 to 300 bp), almost as soon as enough DNA has emerged from the replisome to allow the formation of nucleosome core particles (52). A key protein involved in coupling nucleosome assembly to DNA replication is chromatin assembly factor 1 (CAF-1). CAF-1 is a complex of three polypeptide subunits, known as p150, p60, and RbAp48 in vertebrates, that mediates the first step in nucleosome formation by depositing newly synthesized histone H3/H4 onto DNA (25, 50).In mouse and human cells, CAF-1 localizes to virtually all DNA replication foci throughout the S phase (28, 38, 49, 54). This strongly argues that CAF-1 is a physiologically relevant histone H3/H4 nucleosome assembly factor. In addition, disruption of CAF-1 function in human cells results in a severe loss of viability that is accompanied by spontaneous DNA damage and a block in S-phase progression (20, 40, 60). Thus, unlike in Saccharomyces cerevisiae, the function of CAF-1 in vertebrates cannot be replaced by that of other nucleosome factors, such as members of the Hir protein family or Rtt106 (24, 27, 29). This may be because, unlike CAF-1, HIRA (a human homologue of yeast Hir1 and Hir2) does not associate with core histones that are synthesized during S phase (55). In human cells, the ability to promote nucleosome assembly preferentially onto replicating DNA is thus far unique to CAF-1.This distinctive property of CAF-1 is mediated through proliferating cell nuclear antigen (PCNA), a homotrimeric ring that encircles double-stranded DNA (4) and acts as a sliding clamp to tether DNA polymerases to their DNA substrate and thereby enhance their processivity. Several lines of biochemical and genetic evidence support the role of PCNA in CAF-1-mediated nucleosome assembly. First, CAF-1 colocalizes with PCNA in vivo and binds directly to PCNA in vitro (27, 35, 49, 61). Second, even in the presence of excess unreplicated DNA, CAF-1 can select fully replicated plasmid DNA molecules as preferential substrates for histone deposition, but only when those molecules are associated with PCNA (49). Third, PCNA-driven DNA synthesis can also attract CAF-1 to sites of DNA repair events, such as nucleotide excision repair (12, 15, 32, 35). Fourth, a specific PCNA mutation impairs the role of CAF-1 in telomeric silencing in S. cerevisiae (48, 61). Interestingly, a number of PCNA mutations that reduce its interaction with other PCNA-binding proteins have apparently no effect on CAF-1 function in vivo (48, 61). This implies that the interaction of CAF-1 with PCNA is substantially different from that of other PCNA-binding proteins.Enhancing DNA polymerase processivity is not the only function of PCNA in DNA replication. The sliding clamp also directly binds to other replication enzymes, such as DNA ligase 1, DNA polymerase δ, and FEN1 (14, 21, 37). In addition to its roles in DNA synthesis and nucleosome assembly, PCNA also directly binds to a number of enzymes that continuously monitor and correct the quality of nascent DNA. These include enzymes involved in epigenetic inheritance, such as the maintenance DNA methyltransferase DNMT1 (8), base excision repair (UNG2) (42), mismatch repair (MSH3 and MSH6) (9), DNA lesion bypass (23), and many other processes (31, 36). Even subtle defects in many of these processes, including CAF-1-dependent nucleosome assembly (39), lead to either chromosome rearrangements or mutator phenotypes, which are common features of many human cancers. Surprisingly, many of these enzymes interact with PCNA via canonical PCNA interaction peptides (PIPs) that conform to the consensus sequence QXXhXXaa, where Q is a glutamine, h is a hydrophobic residue (valine, methionine, leucine, or isoleucine), a is an aromatic residue (phenylalanine, tyrosine, tryptophan, or occasionally histidine), and X represents any amino acid. Therefore, regulatory mechanisms must exist to ensure that these fundamentally distinct PCNA-dependent processes occur in a carefully orchestrated manner without mutually interfering with each other.In order to understand how the action of CAF-1 is coordinated with that of other PCNA-binding proteins at replication forks, we carried out a thorough study of CAF-1 PIPs by analyzing their functions using a number of assays. We found that the p150 subunit of CAF-1 contains two fundamentally distinct PIPs. The N-terminal motif (PIP1) binds strongly to PCNA in vitro but is dispensable for nucleosome assembly during simian virus 40 (SV40) DNA replication. In contrast, despite the lack of a key conserved residue, the second PIP (PIP2) of CAF-1 is crucial for replication-dependent nucleosome assembly in vitro and for targeting CAF-1 to DNA replication foci in vivo. Remarkably, although PIP2 exhibits some features of canonical PIPs, it binds only weakly to PCNA in vitro. We suggest that regulated PCNA binding via this peptide may play an important role in ensuring that CAF-1 can efficiently deposit histones behind replication forks without competing with the numerous other enzymes that require continuous access to PCNA during DNA replication. Consistent with this, we show that CAF-1 PIPs possess the ability to preferentially interfere with nucleosome assembly rather than with DNA synthesis.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

The ESCRT-Associated Protein Alix Recruits the Ubiquitin Ligase Nedd4-1 To Facilitate HIV-1 Release through the LYPXnL L Domain Motif

The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPX_nL, that bind Tsg101 and Alix, respectively. Interactions with these two cellular proteins recruit members of the host''s fission machinery (ESCRT) to facilitate HIV-1 release. Other retroviruses gain access to the host ESCRT components by utilizing a PPXY-type L domain that interacts with cellular Nedd4-like ubiquitin ligases. Despite the absence of a PPXY motif in HIV-1 Gag, interaction with the ubiquitin ligase Nedd4-2 was recently shown to stimulate HIV-1 release. We show here that another Nedd4-like ubiquitin ligase, Nedd4-1, corrected release defects resulting from the disruption of PTAP (PTAP⁻), suggesting that HIV-1 Gag also recruits Nedd4-1 to facilitate virus release. Notably, Nedd4-1 remediation of HIV-1 PTAP⁻ budding defects is independent of cellular Tsg101, implying that Nedd4-1''s function in HIV-1 release does not involve ESCRT-I components and is therefore distinct from that of Nedd4-2. Consistent with this finding, deletion of the p6 region decreased Nedd4-1-Gag interaction, and disruption of the LYPX_nL motif eliminated Nedd4-1-mediated restoration of HIV-1 PTAP⁻. This result indicated that both Nedd4-1 interaction with Gag and function in virus release occur through the Alix-binding LYPX_nL motif. Mutations of basic residues located in the NC domain of Gag that are critical for Alix''s facilitation of HIV-1 release, also disrupted release mediated by Nedd4-1, further confirming a Nedd4-1-Alix functional interdependence. In fact we found that Nedd4-1 binds Alix in both immunoprecipitation and yeast-two-hybrid assays. In addition, Nedd4-1 requires its catalytic activity to promote virus release. Remarkably, RNAi knockdown of cellular Nedd4-1 eliminated Alix ubiquitination in the cell and impeded its ability to function in HIV-1 release. Together our data support a model in which Alix recruits Nedd4-1 to facilitate HIV-1 release mediated through the LYPX_nL/Alix budding pathway via a mechanism that involves Alix ubiquitination.Retroviral Gag polyproteins bear short conserved sequences that control virus budding and release. As such, these motifs have been dubbed late or L domains (49). Three types of L domains have thus far been characterized: PT/SAP, LYPX_nL, and PPPY motifs (5, 9, 32). They recruit host proteins known to function in the vacuolar protein sorting (vps) of cargo proteins and the generation of multivesicular bodies (MVB) compartments (2). It is currently accepted that budding of vesicles into MVB involves the sequential recruitment of endosomal sorting complexes required for transport (ESCRT-I, -II, and -III) and the activity of the VPS4 AAA-ATPase (22). These sorting events are believed to be triggered by recognition of ubiquitin molecules conjugated to cargo proteins (20, 24, 41). For retrovirus budding, L domain motifs are the primary signals in Gag that elicit the recruitment of ESCRT components to facilitate viral budding. Consequently, mutations in L domain motifs or dominant-negative interference with the function of ESCRT-III members or the VPS4 ATPase adversely affect virus release. This indicates that Gag interactions with the ESCRT machinery are necessary for virus budding and separation from the cell (7, 10, 15, 16, 21, 28, 44).Two late domains have been identified within the p6 region of human immunodeficiency virus type 1 (HIV-1) Gag protein: the PTAP and LYPX_nL motifs. The PTAP motif binds the cellular protein Tsg101 (15, 39, 40, 47), whereas the LYPX_nL motif is the docking site for Alix (44). Tsg101 functions in HIV-1 budding (15) as a member of ESCRT-I (30, 48), a soluble complex required for the generation of MVB. This process is topologically similar to HIV-1 budding and requires the recruitment of ESCRT-III members called the charged-multivesicular body proteins (3, 29, 48) and the activity of the VPS4 AAA-ATPase (4, 48). In addition to binding the LYPX_nL motif, Alix also interacts with the nucleocapsid (NC) domain of HIV-1 Gag (13, 38), thus linking Gag to components of ESCRT-III that are critical for virus release (13).Other retroviruses, including the human T-cell leukemia virus (HTLV) and the Moloney murine leukemia virus (MoMLV), utilize the PPPY-type L domain to efficiently release virus (7, 26, 51). The PPPY motif binds members of the Nedd4-like ubiquitin ligase family (6, 7, 16, 19, 25, 43), whose normal cellular function is to ubiquitinate cargo proteins and target them into the MVB sorting pathway (11, 12, 20). Members of the Nedd4-like ubiquitin ligase family include Nedd4-1, Nedd4-2 (also known as Nedd4L), WWP-1/2, and Itch. They contain three distinct domains: an N-terminal membrane binding C2 domain (12), a central PPPY-interacting WW domain (43), and a C-terminal HECT domain that contains the ubiquitin ligase active site (42). The functional requirement for the binding of Nedd4-like ubiquitin ligases to the PPPY motif in virus budding has been demonstrated (7, 16, 18, 19, 25, 26, 28, 50, 51). Overexpression of dominant-negative mutants of Nedd4-like ligases, ESCRT-III components, or VPS4 cause a potent inhibition of PPPY-dependent virus release (7, 19, 29, 31, 52) and induce assembly and budding defects similar to those observed after perturbation of the PPPY motif (26, 51). These observations demonstrated that Nedd4-like ligases connect Gag encoding PPPY motif to ESCRT-III and VPS4 proteins to facilitate virus release.Whereas the role of Nedd4-like ubiquitin ligases in virus budding has been established, the protein interactions that link them to the cell''s ESCRT-III pathway are still unknown. Evidence for associations of Nedd4-like ligases with ESCRT proteins have been previously reported and include: the binding of Nedd4-like ubiquitin ligases LD1 and Nedd4-1 to ESCRT-I member Tsg101 (6, 31), the colocalization of multiple Nedd4-like ubiquitin ligases with endosomal compartments (1, 28), the requirement of the cell''s ESCRT pathway for Itch mediated L domain independent stimulation of MoMLV release (23), and the ubiquitination of ESCRT-I components with a shorter isoform, Nedd4-2s (8). Therefore, Nedd4-like ubiquitin ligase interactions with members of the cell''s ESCRT pathway may provide retroviral Gag with access to the host budding machinery required for virus release.Although HIV-1 Gag does not carry the PPPY canonical sequence known to interact with Nedd4-like ubiquitin ligases, both Nedd4-1 and Nedd4-2 were shown to restore the release of the HIV-1 PTAP⁻ mutant, albeit Nedd4-1 with less efficiency than Nedd4-2 (8, 46). These findings suggested that HIV-1 might utilize cellular Nedd4-like ubiquitin ligases to increase virus release. We present here evidence demonstrating that Nedd4-1 interacts with Gag and enhances HIV-1 PTAP⁻ virus release. Furthermore, we show that Nedd4-1''s function in HIV-1 release is distinct from that of Nedd4-2 in both its viral and cellular requirements. Notably, we found that Nedd4-1 enhancement of HIV-1 release requires the Alix-binding LYPX_nL L domain motif in the p6 region and basic residues in the NC domain. In addition, Alix''s facilitation of HIV-1 release requires cellular Nedd4-1, since mutations in NC that prevented Alix-mediated HIV-1 release also eliminated release by overexpression of Nedd4-1. This suggested a Nedd4-1-Alix physical and functional interdependence. In agreement with this, we found Nedd4-1 to bind and ubiquitinate Alix in the cell. Taken together, these results support a model in which Alix recruits Nedd4-1 to facilitate late steps of HIV-1 release through the LYPX_nL L domain motif via a mechanism that involves Alix ubiquitination.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Cyclin A2-Cyclin-Dependent Kinase 2 Cooperates with the PLK1-SCFβ-TrCP1-EMI1-Anaphase-Promoting Complex/Cyclosome Axis To Promote Genome Reduplication in the Absence of Mitosis

Limiting genome replication to once per cell cycle is vital for maintaining genome stability. Inhibition of cyclin-dependent kinase 1 (CDK1) with the specific inhibitor RO3306 is sufficient to trigger multiple rounds of genome reduplication. We demonstrated that although anaphase-promoting complex/cyclosome (APC/C) remained inactive during the initial G₂ arrest, it was activated upon prolonged inhibition of CDK1. Using cellular biosensors and live-cell imaging, we provide direct evidence that genome reduplication was associated with oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis at the single-cell level. Genome reduplication was abolished by ectopic expression of EMI1 or depletion of CDC20 or CDH1, suggesting the critical role of the EMI1-APC/C axis. In support of this, degradation of EMI1 itself and genome reduplication were delayed after downregulation of PLK1 and β-TrCP1. In the absence of CDK1 activity, activation of APC/C and genome reduplication was dependent on cyclin A2 and CDK2. Genome reduplication was then promoted by a combination of APC/C-dependent destruction of geminin (thus releasing CDT1), accumulation of cyclin E2-CDK2, and CDC6. Collectively, these results underscore the crucial role of cyclin A2-CDK2 in regulating the PLK1-SCF^β-TrCP1-EMI1-APC/C axis and CDC6 to trigger genome reduplication after the activity of CDK1 is suppressed.Limiting genome replication to once per cell cycle is critical for maintaining genome stability and suppressing tumorigenesis (reviewed in reference 18). DNA replication is a biphasic process consisting of origin licensing and origin firing. During late mitosis to early G₁ phase, origins are licensed by orderly loading of prereplicative complex components, including ORC, CDT1, CDC6, and MCM2-7 (reviewed in reference 3). Origin licensing occurs in a biochemical environment characterized by low activity of cyclin-cyclin-dependent kinase (CDK) and high activity of anaphase-promoting complex/cyclosome (APC/C) (reviewed in reference 51). Firing of the origins is coordinated by phosphorylation carried out by cyclin-CDK and DBF4-CDC7 and the binding of other replication factors, including CDC45, MCM10, RPA, and DNA polymerase (reviewed in reference 58).Critical roles in replication have been attributed to both cyclin A and cyclin E, but the distinct roles performed by the two cyclins remain incompletely understood. Cyclin A is especially interesting among the cyclins because of its association with multiple CDKs (CDK1 and CDK2) and its proposed functions in multiple points of the cell cycle (S phase and mitosis). In S phase, cyclin A is believed to be involved in the loading of CDC45 onto origins. Cyclin A is also involved in blocking the reloading of fired origins (reviewed in reference 43). CDT1 is targeted to degradation after phosphorylation by cyclin-CDK in SCF^SKP2- or CUL4-DDB1-mediated mechanisms (15). Geminin accumulates during S phase and inactivates the remaining CDT1 (32). Moreover, CDC6 is phosphorylated by cyclin-CDK and translocated out of the nucleus. Finally, ORC1 (the largest subunit of the ORC) is inactivated either by polyubiquitination by SCF^SKP2 and degradation by the proteasome (38) or by monoubiquitination and dissociation from the chromatin (29). Thus, the high cyclin-CDK and low APC/C activities during S phase prevent the formation of the prereplicative complex and reduplication. The system is reset during the next mitosis, when APC/C is activated and degrades cyclins and geminin, allowing the prereplicative complex to form again.Genome reduplication generates polyploid cells. A growing body of evidence indicates that polyploidization can initiate chromosomal instability and aneuploidy. A seminal study by Fujiwara et al. (16) indicates that tetraploids can be generated by transient blocking of cytokinesis in p53-null mouse mammary epithelial cells. Importantly, tetraploidization promotes aneuploidy and tumorigenesis (16). Another study reported that chromosome nondisjunction (both copies of a chromosome segregate to the same daughter cells) leads to binucleated tetraploids by promoting cleavage furrow regression; the tetraploid cells then become aneuploidy through further divisions (48). This and other studies provide strong evidence of the importance of tetraploidization as an early step in tumorigenesis (reviewed in reference 53).While rereplication is stringently prevented in the normal cell cycle, multiple rounds of genome reduplication, called endoreduplication, occur in cell types such as megakaryocytes, trophoblast giant cells, numerous plant cells (26), and in the salivary glands of Drosophila melanogaster (49). In yeasts, different levels of a single CDK are believed to allow origin licensing and firing and prevent relicensing and mitosis (reviewed in reference 45). In contrast, the complex interplay between different cyclin-CDK complexes and licensing factors to prevent genome reduplication in higher eukaryotes remains to be fully characterized.Cyclin E is required for the endoreduplication cycles in Drosophila cells (14, 31, 57), trophoblasts, and megakaryocytes (20, 40). In fact, ectopic expression of cyclin E can promote endoreduplication in megakaryocytes (19). In contrast, although a decrease in cyclin A promotes endoreduplication in plant cells (24, 62) and Drosophila cells (39), it does not appear to be the case for megakaryocytic cell lines (19, 65, 66). A decrease in cyclin B1, but not cyclin A2, has been reported to be required for endoreduplication in megakaryocytes (66). Precisely which cyclin-CDK complexes are involved in safeguarding rereplication remains largely unresolved. Rereplication induced by EMI1 depletion is correlated with a reduction of cyclin A2 and cyclin B1, which can be rescued with nondestructible cyclin A2 (34). However, rereplication in HeLa cells is induced only weakly by cyclin A2 depletion, but it occurs more efficiently after codepletion of geminin (34). Conversely, expression of cyclin A2 (but not cyclin E) potentiates the rereplication induced by CDC6 and CDT1 in mammalian cells (56).In contrast to the complexity and uncertainty about the different cyclins in DNA reduplication, the central role of inactivation of mitotic CDK1 is generally accepted. This has been observed in a wide range of endoreduplication cycles, including those in maize endosperm (21), Drosophila cells (49), trophoblasts (54), and megakaryocytes (65). Likewise, extensive DNA reduplication can be triggered by disruption of CDK1 in a mammalian cell line (7, 25, 28). The molecular basis of how CDK1 inactivation contributes to genome reduplication remains to be defined.The prevailing view is that APC/C plays a salient role in preventing rereplication. The orthologs of CDH1 in plant cells (Ccs52A) and in Drosophila cells (fzr) are both essential for endoreduplication (26, 49). Unscheduled activation of APC/C reduces the concentrations of mitotic cyclins and geminin, resulting in rereplication (12, 34). An extra level of regulation provided by the APC/C inhibitor EMI1 has been uncovered recently. EMI1 begins to accumulate at the G₁/S transition, thereby inactivating APC/C and allowing the accumulation of cyclins and geminin (46). Accordingly, depletion of EMI1 with RNA interference promotes unscheduled APC/C activation and rereplication (12, 34).In this study, we utilized a specific CDK1 chemical inhibitor to induce whole-genome reduplication in cancer cells. We found that genome reduplication was associated with spontaneous oscillation of APC/C activity and nuclear-cytoplasmic shuttling of CDC6 even in the absence of mitosis. Moreover, the PLK1-SCF^β-TrCP1-EMI1 axis and cyclin A2-CDK2 were inextricably linked to the APC/C activation and genome reduplication. These data extended our understanding of the role of the different cyclin-CDK complexes in coordinating genome reduplication.     

Lipid Phosphate Phosphatase 3 Stabilization of β-Catenin Induces Endothelial Cell Migration and Formation of Branching Point Structures

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures.Angiogenesis, the formation of new blood vessels, involves several well-coordinated cellular processes, including endothelial cell (EC) migration, synthesis and deposition of extracellular matrix proteins, such as fibronectin, cell-cell adhesion, and formation of branching point structures (1-3, 19, 33); however, less is known about the underlying mechanisms of these processes (6, 8, 12, 14, 16, 17). For example, adherens junctions (AJs), which mediate cell-cell adhesion between ECs, may be involved in limiting the extent of cell migration (2, 14, 38, 40). VE-cadherin, a protein found in AJs, is a single-pass transmembrane polypeptide responsible for calcium-dependent homophilic interactions through its extracellular domains (2, 38, 40). The VE-cadherin cytoplasmic domain interacts with the Armadillo domain-containing proteins, β-catenin, γ-catenin (plakoglobin), and p120-catenin (p120ctn) (2, 15, 38, 40, 43). Genetic and biochemical evidence documents a crucial role of β-catenin in regulating cell adhesion as well as proliferation secondary to the central position of β-catenin in the Wnt signaling pathway (13, 16, 25, 31, 44). In addition, the juxtamembrane protein p120ctn regulates AJ stability via binding to VE-cadherin (2, 7, 9, 15, 21, 28, 32, 43). The absence of regulation or inappropriate regulation of β-catenin and VE-cadherin functions is linked to cardiovascular disease and tumor progression (2, 6).We previously identified lipid phosphate phosphatase 3 (LPP3), also known as phosphatidic acid phosphatase 2b (PAP2b), in a functional assay of angiogenesis (18, 19, 41, 42). LPP3 not only exhibits lipid phosphatase activity but also functions as a cell-associated integrin ligand (18, 19, 35, 41, 42). The known LPPs (LPP1, LPP2, and LPP3) (20-23) are six transmembrane domain-containing plasma membrane-bound enzymes that dephosphorylate sphingosine-1-phosphate (S1P) and its structural homologues, and thus, these phosphatases generate lipid mediators (4, 5, 23, 35, 39). All LPPs, which contain a single N-glycosylation site and a putative lipid phosphatase motif, are situated such that their N and C termini are within the cell (4, 5, 22, 23, 35, 39). Only the LPP3 isoform contains an Arg-Gly-Asp (RGD) sequence in the second extracellular loop, and this RGD sequence enables LPP3 to bind integrins (18, 19, 22). Transfection experiments with green fluorescent protein (GFP)-tagged LPP1 and LPP3 showed that LPP1 is apically sorted, whereas LPP3 colocalized with E-cadherin at cell-cell contact sites with other Madin-Darby canine kidney (MDCK) cells (22). Mutagenesis and domain swapping experiments established that LPP1 contains an apical targeting signal sequence (FDKTRL) in its N-terminal segment. In contrast, LPP3 contains a dityrosine (109Y/110Y) basolateral sorting motif (22). Interestingly, conventional deletion of Lpp3 is embryonic lethal, since the Lpp3 gene plays a critical role in extraembryonic vasculogenesis independent of its lipid phosphatase activity (11). In addition, an LPP3-neutralizing antibody was shown to prevent cell-cell interactions (19, 42) and angiogenesis (42). Here, we addressed the hypothesis that LPP3 plays a key role in EC migration, cell-cell adhesion, and formation of branching point structures by stimulating β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) signaling.